Biosensor and method of analyte measuring

ABSTRACT

A biosensor capable of analyzing an object, such as antigen, antibody, DNA or RNA, through detection of magnetic field to thereby allow washout of unbound label molecules to be unnecessary, which biosensor is compact and available at low price, excelling in detection precision. Coils are arranged at an upper part and a lower part of a magnetic sensor using a hall element as a magnetic field detection element. An object and magnetic particles having an antibody capable of specific bonding with the object bound to the surface thereof are introduced in the magnetic sensor having a molecular receptor capable of specific bonding with the object attached to the surface thereof. Therefore, a change in magnetic field by magnetic particles bonded through the molecular receptor to the surface of the magnetic sensor is detected by means of the hall element. At that time, one applied magnetic field is set so that the magnetization intensity of magnetic particles falls within the range from initial magnetic permeability to maximum magnetic permeability while another applied magnetic field is set so that the magnetization intensity of some or all of the magnetic particles becomes saturated, and output signals are compared with each other. Thus, the amount of bonded magnetic particles can be identified.

TECHNICAL FIELD

The present invention relates to a sensor for measuring an amount ofmagnetic particles and a method of measuring the same, and in particularto a biosensor using magnetic particles and a method of assaying anobject.

BACKGROUND ART

Recently, in clinical diagnosis/detection and analysis of the genes,immunological methods utilizing a specific interaction between specificmolecules, such as an antigen and antibody thereto, and the like, areused to detect antigen, antibody, DNA (Deoxyribonucleic Acid), RNA(Ribonucleic Acid) and the like.

One of these methods, a solid phase binding assay includes a methodusing magnetic particles. FIG. 15 shows a schematic diagram of a solidphase assay using conventional magnetic particles.

As shown in the figure, the assay for an object 94 is carried out usinga solid phase 91, a molecular receptor 95, a magnetic particle 92 and asecondary molecular receptor 93.

The solid phase 91 has a surface of the solid phase in contact with thesample solution and the molecular receptors 95 are immobilized thereto.Polystyrene beads, walls of a reaction vessel, substrates and the likeare used as the solid phase 91.

The molecular receptor 95 is a substance that specifically binds to theobject 94, such as antigen, antibody, DNA, RNA or the like, which existsin the sample solution. The molecular receptor 95 is such a molecule asantigen, antibody, DNA, RNA or the like, which can specifically bind tothe object 94.

The magnetic particle 92 is a particle having magnetization and used asa labeling material. That is, by detecting the magnetic field formed bythe magnetic particle 92, the amount of the magnetic particle 92 isdetermined, and the presence or absence, or concentration of an object94 in a sample solution is determined. In addition to the magneticparticles 92, a substance that emit detectable signals, such asradioactive, luminescent and chemiluminescent materials, enzymes and thelike, maybe used as a label. The known assays using these labels includeenzyme immunoassay (EIA) using antigen-antibody reaction, andchemiluminescent assays, such as a strictly defined chemiluminescentassay (CLIA) using an immunoassayed compound labeled with achemiluminescent material, chemiluminescent enzyme immunoassay (CLEIA)in which enzyme activity is detected at a high sensitivity by using achemiluminescent compound in the detection system, and the like.

The secondary molecular receptor 93 that is previously immobilized tothe magnetic surface is an antibody that binds specifically to theobject 94.

In the analysis shown in FIG. 15, firstly a test solution containing theobject 94 is added to the solid phase 91 to which the molecular receptor95 is immobilized beforehand. By this procedure the object 94 bindsspecifically. Other substances in the sample solution stay in thesolution without binding to the solid phase 91. Next, the magneticparticles 92, on which the secondary molecular receptors 93 areimmobilized, are added into the sample solution. By this procedure, thesecondary molecular receptor 93 binds specifically to the object 94 thatis bound specifically to the molecular receptor 95 immobilized onto thesolid phase 91. Then, the magnetic particles 92 bound onto the solidphase is quantitated by detecting the magnetization of the magneticparticles 92. By this procedure, the concentration or the location ofthe object 94 bound onto the solid phase may be determined. The methodsfor detecting the magnetization using magnetoresistance elementsdisposed in array form are disclosed in the patent documents 1 and 2.

Further, the assays using these labels include sandwich assay in whichthe object bound specifically to the molecular receptor described aboveis bound specifically to another molecule label, and also competitiveassay, in which the object and a different molecule label competes tobind to the molecular receptor.

Thus, in the conventional methods, signals from the label, such as lightemission and the like, are detected by a device, such as an opticaldetection device and the like capable of detecting these signals. Inthese methods, it is necessary to capture signals only from the label ofthe molecule bound specifically to another molecule immobilized onto thesolid phase. However, in the optical detection method, accurate analysesmay not be carried out in the presence of unbound labeled moleculesbecause the signals from these labels are also captured. Therefore, itis necessary to washout the unbound labeled molecules completely.Further, in the optical detection device, very weak optical signals needto be detected, which creates difficulty in making the device compactand low cost.

On the other hand, as disclosed in the patent document 1, it is notnecessary to washout unbound labeled molecules in the method ofdetecting by the magnetoresistance element using the magnetic particlesas the label. However, in a detection chip on which themagnetoresistance elements are disposed in array form, a switchingcircuit is needed to output the signals from each element independently.Electric interconnects are required from each element disposed in arrayform to the switching circuit. Therefore, this creates problems, such asdifficulty in making compact and the like, because as the number of theelements are increased, the interconnects are more complicated and themore area is needed to accommodate the interconnects.

Similarly, in the detection device that detects magnetic flux in thepatent document 2 described above, the detection circuit for themagnetic particles includes a bridge circuit composed of themagnetoresistance elements and transistors serving as switchingelements. However, since the magnetoresistance element requires magneticmaterial, the steps for formation and processing of the thin magneticfilm have to be carried out after a part of the circuit including thetransistors is processed by a general production process for integratedcircuits.

Further, the patent document 3 discloses the method for detectingmagnetic particles, as in the patent document 1, by disposing the hallelements in an array-like formation.

However, in the patent document 3, the output signals of the hallelements to which the magnetic particles are not bound must be used as astandard for the output signals of the hall elements to which themagnetic particles are bound. Furthermore, since the output signal fromthe hall element to which the magnetic particles are bound is so smallthat the detection becomes difficult when the size of the magneticparticles is smaller than the size of the hall element.

The objective of the present invention is to provide a magnetic sensor,which is compact, inexpensive and with a higher detection sensitivity,and a method of measuring the amount of the magnetic particles. Further,applying this sensor and the method of measuring, the present inventionprovides a biosensor, which is compact, inexpensive and with higherdetection sensitivity, and an assay method to analyze the objects, suchas antigen, antibody, DNA, RNA and the like, by detecting the magnetismusing the magnetic particles as a label, without the washout of theunbound labeling molecules.

Patent Document 1: U.S. Pat. No. 5,981,297, Description.

Patent Document 2: International Publication WO97/45740, Pamphlet.

Patent Document 3: International Publication WO03/67258, Pamphlet.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided the sensorincluding a magnetic sensor composed of a plurality of magnetic fielddetection elements arranged two dimensionally in X rows and Y columns (Xand Y are natural numbers), which generate the output values accordingto the intensity of the detected magnetic field, and measuring an amountof magnetic particles present on the aforementioned magnetic sensorbased on the output values, characterized in that the sensor comprises asignal processing means that determines the amount of the magneticparticles based on the dispersion of the output value distribution,which is obtained from the output value of a plurality of the magneticfield detection elements.

When an external magnetic field is applied to the magnetic sensor in astate where the magnetic particles are not bound to the magnetic sensor,there is no variation of output values of a plurality of magnetic fielddetector elements (in the case of the ideal condition that there is novariation of sensitivity of each magnetic field detection element).However, when the magnetic particles are bound to this magnetic sensor,the magnetic particles bound to the magnetic sensor cause localdisturbance of the magnetic field applied to the magnetic sensor, whichresults in variation of output values of a plurality of magnetic fielddetector elements. Since the degree of this variation is dependent onthe amount of bound magnetic particles, the bound magnetic particles canbe quantified based thereon.

For evaluation of this degree of variation, it is preferable to usedispersion indices such as standard deviation, average deviation,variance and the like.

Further, the output value of the magnetic field detection elementincludes the output value, or values corresponding to intensity ofmagnetic field, wherein the values are obtained in the output values.

In further embodiment, the biosensor of the present invention is theaforementioned sensor and the magnetic particles bind to an object whichbinds to the magnetic sensor, and the biosensor of the present inventionis characterized in that the signal processing means determines anamount of magnetic particles bound to the aforementioned magnetic sensorthrough the object and determines an amount of the object based on theamount of the magnetic particles.

In further embodiments, the biosensor of the present invention ischaracterized in that the magnetic particles bind specifically with theobject that binds to the aforementioned magnetic sensor, and that theamount of these magnetic particles bound to the aforementioned magneticsensor through the aforementioned object is determined.

In further embodiments, the biosensor of the present invention ischaracterized in that the aforementioned signal processing meansdetermines the amount of the aforementioned bound magnetic particlesbased on the differential between the dispersion of the aforementionedoutput value distribution and the dispersion of the referencedistribution obtained from the output value of a plurality of theaforementioned magnetic field detection elements in the state where themagnetic particles are not bound to the aforementioned magnetic sensor.

In such a way, when the sensitivity of each magnetic field detectionelement is nonuniform, an accurate measurement may be made by comparingthe dispersion of the reference distribution with the dispersion of theoutput values distribution, after obtaining the reference distributionin the state where the magnetic particles are not bound to the magneticsensor.

In further embodiments, the biosensor of the present invention includesa means for applying external magnetic field of greatly differentintensities to the aforementioned magnetic sensor bound to the magneticparticles, wherein one of the aforementioned external magnetic field ofgreatly different intensities is such a strong magnetic field that themagnetization of at least some of the bound magnetic particles becomessaturated, and another external magnetic field is such a weak magneticfield that the aforementioned bound magnetic particles each have amagnetic permeability falling within a range from an initial magneticpermeability to a maximum magnetic permeability, and is characterized inthat the aforementioned signal processing means determines an amount ofthe aforementioned bound magnetic particles based on difference betweenthe dispersion of the aforementioned output value distribution when theaforementioned strong magnetic field is applied and the dispersion ofthe aforementioned output value distribution when the aforementionedweak magnetic field is applied.

Here, the output values of the magnetic field detection element includethe output value, or, based on the values obtained when the strongmagnetic field and weak magnetic field are applied, values correspondingto intensity of magnetic field in each case.

In further specific embodiments, the present invention provides thebiosensor that includes a magnetic sensor composed of a plurality ofmagnetic field detection elements disposed two dimensionally in X rowsand Y columns (X and Y are natural numbers), which generate outputvalues according to intensity of detected magnetic field, and thatmeasures an amount of magnetic particles bound to the magnetic sensorbased on the output values. In further embodiments, a biosensor of thepresent invention including: a means for applying external magneticfield of greatly different intensities to the magnetic sensor bound tothe magnetic particles; a signal processing means that determines theamount of the bound magnetic particles by comparing output values ofeach of the magnetic field detection element when the external magneticfield of greatly different intensities is applied, is characterized inthat one of the external magnetic field of greatly different intensitiesis such a strong magnetic field that the magnetization of at least someof the bound magnetic particles becomes saturated, and another externalmagnetic field is such a weak magnetic field that the bound magneticparticles each have a magnetic permeability falling within a range froman initial magnetic permeability to a maximum magnetic permeability.

In further embodiments, a biosensor of the present invention ischaracterized in that the strong magnetic field changes intensity withinsuch a range that the magnetization of at least some of the boundmagnetic particles becomes saturated, and the weak magnetic fieldchanges intensity within such a range that the permeability of themagnetic particles changes from initial magnetic permeability to maximummagnetic permeability, and that the signal processing means obtains froma plurality of magnetic field detection elements changes in outputvalues according to changes in intensity of external magnetic field,when external magnetic fields of greatly different intensities includingthe strong magnetic field and the weak magnetic field are applied, anddetermines the amount of the bound magnetic particles based ondifferentials of the changes in these output values.

This is based on the discovery by the present inventors that whenexternal magnetic field, in which the magnetic permeability of themagnetic particle is between initial and maximum state, is applied, thechange rate of the magnetic flux density at the detection elementagainst the external magnetic field intensity becomes greater becausemagnetization of the magnetic particles is increased in proportion tothe intensity of the external magnetic field, but when the externalmagnetic field intensity is increased further, the change rate of themagnetic flux density is getting smaller because the magnetization ofthe magnetic particles become saturated.

Below, a description is given using FIG. 1A. As shown in FIG. 1A, whenan external magnetic field with a magnetic flux density B is applied inthe absence of magnetic particle 51, the magnetic flux density detectedby hall elements 2, magnetic field detection elements, is the same asthe external magnetic field, B. In the condition that the magneticparticle 51 are bound, the magnetic flux density detected by the hallelements 2 is changed because the magnetic particle 51 are magnetized bythe external magnetic field. Here, the external magnetic field isapplied vertically to the magnetosensitive layer of the hall element 2.

FIG. 1B shows a relationship between the change rate of magnetic fluxdensity against the change in intensity of the external magnetic fieldand distance from the bound magnetic particles. FIG. 1B is a graphshowing the change rate of magnetic flux density to the externalmagnetic field with the magnetic flux density B at the position of thedotted line a in FIG. 1A, and the vertical axis represents the changerate of the magnetic flux density and the horizontal axis represents theposition corresponding to the arrangement of the hall elements in FIG.1A. Further, the solid line L1 represents the change rate of themagnetic flux density when a weak external magnetic field is applied sothat the magnetic permeability of the magnetic particle 51 falls betweenthe initial magnetic permeability and the maximum permeability (weakmagnetic field), and the dotted line L2 represents the change rate ofthe magnetic flux density when a strong magnetic field is applied sothat the magnetization intensity of some or all of the magneticparticles becomes saturated (strong magnetic field).

Since the magnetization of the magnetic particle 51 is proportional tothe external magnetic field between the initial magnetic permeabilityand the maximum magnetic permeability, the change rate of the magneticflux density is great, but as the intensity of the external magneticfield is increased, the change rate of the magnetic flux density isgetting smaller because the magnetization of the magnetic particle 51becomes saturated.

Also, directly under the bound magnetic particle 51 (the location ofhall element 2 a), the magnetic flux density is increased due to themagnetization of the magnetic particle 51, but at the location a littleaway from the directly under the magnetic particle 51 (the location ofhall element 2 b), the magnetic flux density is decreased due to themagnetization of the magnetic particle 51.

Further, when a strong external magnetic field is applied, the changerate of the magnetic flux density at the locations of the hall elements2 a and 2 b is decreased in absolute value, although the change rate maybe plus or minus, compared with that occurring when a weak externalmagnetic field is applied. The change rate of the magnetic flux at thelocation away from the magnetic particle 51 (the location of hallelement 2 c) is not affected by the intensity of the external magneticfield.

Therefore, by comparing the change rates of the magnetic flux densityunder a weak external magnetic field, where the magnetic permeability ofthe magnetic particles falls between the initial magnetic permeabilityand the maximum permeability with that under a strong external magneticfield, where magnetization of a part or all of the magnetic particles issaturated, for example, by judging whether they are different or notbased on the output of the magnetic field detection elements, it can bedetermined whether the magnetic particles are bound near the magneticfield detection element or not. In the present invention, an amount ofchange in the output values (output change rate) of the magnetic fielddetection elements against the change in intensity of the externalmagnetic field is used as a value corresponding to the change rate ofthe magnetic flux density against the change in intensity of theexternal magnetic field.

Further, the external magnetic field, either strong or weak magneticfield, may be a DC magnetic field or AC magnetic field. At this time,applying a DC magnetic field so that the magnetization of the magneticparticles is saturated, makes the change rate zero and more accuratemeasurement possible.

FIG. 2A shows the relationship of the output change rates of a hallelement that is a magnetic field detection element in a weak magneticfield (AC magnetic field) and a strong magnetic field (AC magneticfield+DC magnetic field). The vertical axis represents the output changerate of the hall element under application of a weak magnetic field andthe horizontal axis represents the output change rate of the hallelement under application of a strong magnetic field. The component ofthe AC magnetic field in the weak and strong magnetic fields is thesame.

When the magnetic particles are not present on the hall element, andthere is no noise or variation of sensitivity, the output change rate ofthe hall element in the weak magnetic field and in the strong magneticfield is the same, and 1. However, the sensitivities of a plurality ofhall elements fabricated on a same sensor chip by the conventional CMOSprocess are not equal and varied due to the process fluctuation. Thus,the plots of the output change rate of a plurality of hall elementsdistribute to the direction of arrow Y1 on the line with a slope of 1 inFIG. 2A. Even though there is such a variation of the sensitivity amongthe hall elements, the outputs of the same hall element in the weakmagnetic field and in the strong magnetic field are equal, and thereforethere is no need for considering the calibration of the sensitivity, aslong as comparing these outputs.

Next, when a magnetic particle is bound directly on top of a hallelement, as explained in FIGS. 1A and 1B, the magnetic flux isconcentrated in the weak magnetic field by the magnetic particle and theoutput change rate of the hall element is increased. In the strongmagnetic field, the magnetization of the magnetic particle is saturated,and thus the concentrative effect by the alternating current componentis reduced and the output change rate of the hall element does notincrease as much as in the weak magnetic field (if the magnetization iscompletely saturated, the output change rate is not increased).Therefore, the output change rate is distributed to the direction ofarrow Y2 in FIG. 2A.

When the magnetic particle is bound, not directly on the top of the hallelement but at the location a little away from the top, the magneticflux is converged directly under the magnetic particle as explained inFIGS. 1A and 1B and reduced. In this case the output change rate isdistributed to the direction of arrow 3 in FIG. 2A.

Further, since noise is present in the real manufactured hall elements,the output change rate is distributed to all the direction as shown byarrow 4 in FIG. 2A.

Next, an example of the measurements for the output change rate in eachhall element in the weak magnetic field (AC magnetic field) and thestrong magnetic field (AC magnetic field+DC magnetic field) are shownwhen no magnetic particle is bound to any of the hall elements (FIG. 2B)and when the magnetic particles are bound to the surface of the sensorchip (FIG. 2C). The composition of the AC magnetic field in the weak andstrong magnetic fields is the same.

When the magnetic particle is not bound, it is confirmed that the outputchange rates are distributed around the line with a slope 1 as shown inFIG. 2B.

When the magnetic particle is bound, as shown clearly in FIG. 2C, it isconfirmed that the values of the output change rate in the strongmagnetic field in the weak magnetic field are greater and the dispersioncondition of the plots widens to the direction of the vertical axis.

By these procedures, it can be decided whether the output change rate ofthe magnetic field detection element takes a different value or not whenexternal magnetic fields of greatly different intensities are applied.Through such decision the binding (or not binding) of the magneticparticle may be judged, and the amount of the magnetic particles boundto the magnetic sensor may be determined. Thus, even if there arevariations in the sensitivity and the like of each magnetic fielddetection element, accurate measurements may still be carried out.Because of that, operations for calibration of the variation of thesensitivity and the like of each magnetic field detection element, andarrangement of the reference hall elements not bound to the magneticparticles are unnecessary.

In further specific embodiments, the biosensor according to the presentinvention is characterized in that output values are obtained from aplurality of the aforementioned magnetic field detection elements firstapplying the weak magnetic field by the means for applying externalmagnetic field, and then another set of output values are obtained froma plurality of the magnetic field detection elements applying the strongmagnetic field by the means for applying external magnetic field.

By such a procedure, a part of the magnetic particles bound to thesurface of the biosensor is released by the strong magnetic field, andthe signals may be obtained in the condition more similar to that themagnetic particles are not bound, making it possible to measure withhigher accuracy.

In further embodiments, it is characterized in that the aforementionedmeans for applying external magnetic field can apply a DC magneticfield, and still further can apply an AC magnetic field.

Here, the DC magnetic field is a magnetic field with a constantdirection and intensity, and the AC magnetic field is a magnetic fieldwith the direction and intensity that alter periodically, for example,that can be generated by flowing alternating current in a coil.

In further embodiments, it is characterized in that the aforementionedweak magnetic field is an AC magnetic field with such an intensity thata magnetic permeability of the bound magnetic particle falls within arange from initial magnetic permeability to maximum magneticpermeability, and the aforementioned strong magnetic field is the ACmagnetic field added with a DC magnetic field and is an externalmagnetic field with such an intensity that the magnetization of at leastsome of the bound magnetic particles becomes saturated.

In further embodiments, it is characterized in that the aforementionedsignal processing means includes: a noise prediction means that predictsnoise components from frequency components other than thosecorresponding to the AC magnetic field included in output values fromthe magnetic field detection elements; and a noise removal means thatremoves noise components from frequency components corresponding to theAC magnetic field included in output values from the magnetic fielddetection elements based on the noise components predicted by the noiseprediction means.

Next, it is characterized in that magnetic particles bound to themagnetic sensor are associated with other magnetic particles in adirection of the magnetic flux formed by the external magnetic field.

In this way, it becomes possible to measure with higher sensitivitybecause the concentrative effect of magnetic flux is enhanced by themagnetic particles bound to the magnetic sensor, and the signal of themagnetic field detection element is amplified, by binding the magneticparticles, which are bound to the magnetic sensor through object and atthe same time magnetized by the external magnetic field, with anothermagnetic particles, which are not bound to the magnetic sensor throughobject but magnetized by the external magnetic field, with theinteraction of their magnetic force.

In further embodiments, it is characterized in that the aforementionedmagnetic field detection elements generate output values in proportionto a magnetic flux density of a flux formed in a detection space capableof magnetic field detection.

Using the magnetic field detection element which generate the outputvalues in proportion to the magnetic flux density in the detection areain this way, it is possible to increase the accuracy of measurement ofthe magnetic flux density in external magnetic field of greatlydifferent intensities as described above.

In further embodiment, the aforementioned magnetic field detectionelement is characterized by including a hall element.

As described above, the measurement accuracy becomes superior by usingthe hall element which generates output values in proportion to themagnetic flux density in the detection area.

Next, it is characterized by further including a selection means forselecting an arbitrary element among a plurality of said magnetic fielddetection elements and obtaining output values therefrom.

By this procedure, information about 2 dimensional location of themagnetic particles present on the surface of the magnetic sensor may beobtained.

In further embodiments, it is characterized by further including asignal amplification circuit that amplifies output values of themagnetic field detection element selected by the aforementionedselection means and in that the magnetic sensor, the selection means andthis signal amplification circuit are formed on a chip.

By this system, the influence of induced electromotive force byalternating current magnetic field may be reduced. That is, the use ofalternating current magnetic field as an external magnetic fieldgenerates induced electromotive force in interconnects for obtainingsignals from the sensor, and this is added as a noise to the signals. Byincluding the signal amplification circuit, the amplified signals areretrieved, and thus, even if the induced electromotive force by theexternal magnetic field is added, this influence may be reduced.

Further, the magnetic sensor may be made compact and also may be used bydisposing according to the sample solution.

In further embodiments, it is characterized in that the aforementionedhall element includes: a pair of current terminals; a gate electrodethat control a current flowing between the pair of current terminals;and a pair of output terminals that are arranged so that a current flowsin almost vertical direction against the current flowing between thepair of current terminals.

In further embodiments, the aforementioned gate electrode is connectedto a gate electrode interconnect, which is common for said hall elementsarranged in a same row; the aforementioned pair of current terminals areconnected to a pair of current terminal interconnects, which are commonfor said hall elements arranged in a same row; and the aforementionedpair of output terminals are connected to a pair of output terminals,which are common for said hall elements, and it is characterized in thatthe aforementioned selection means selects an arbitrary element from aplurality of hall elements and obtains output value thereof, byselecting one from Y numbers of gate electrode interconnects, a pairfrom X numbers of pairs of current terminal interconnects and a pairfrom X numbers of pairs of output terminal interconnects.

By making common interconnects for each row and each column, theselection of a hall element in an arbitrary location may be carried outeasily, and at the same time the number of interconnect may be reduced.By such a composition, production of a magnetic sensor suitable to anobject becomes easier and also it is possible to make the sensorcompact.

In further embodiments, it is characterized in that in each detectionarea capable of magnetic field detection by the aforementioned magneticfield detection elements, an area of a vertical cross section of amagnetic flux formed on a surface of the magnetic sensor is almost thesame as a maximum cross section area of said magnetic particle.

By this system, the number of magnetic particles bound to the magneticsensor and detected by the magnetic field detection elements is limitedto about 1, and thus it is possible to suppress the variation ofmeasured values due to detecting a plurality of magnetic particles andto improve accuracy of the analysis. Further, since this system is tolimit the number of magnetic particles bound to the magnetic sensorwhich may exist in the detection space, the detection space is, forexample, elongated to the direction of the magnetic flux formation, andthe magnetic field detection elements may be able to detect anothermagnetic particles which are linked in a row along the direction of themagnetic flux formation, attracted by the magnetic particles bound tothe magnetic sensor. This is a favorable case because detectionsensitivity for the magnetic particles bound to the magnetic sensor isenhanced by another magnetic particles.

In further embodiments, it is characterized in that each of theaforementioned magnetic field detection element is arranged with a spaceso that each element detects different magnetic particles.

By this system, it is possible to suppress the interferences such as theneighboring magnetic field detection elements detect the same magneticparticle and the like.

In further embodiments, it is characterized in that surface of theaforementioned magnetic sensor is treated so that molecular receptorscapable of binding to said magnetic particles may be immobilizedthereto.

In further embodiments, it is characterized in that surface of theaforementioned magnetic sensor is treated so that molecular receptorscapable of binding to said magnetic particles may be immobilizedselectively to a specific area.

By this procedure, it is possible to control an amount of bound magneticparticles in the detection space and the like.

In further embodiment, it is characterized in that recessescorresponding to the magnetic particles in size are formed on thesurface of the aforementioned magnetic sensor in the detection spacecapable of magnetic field detection, and that the molecular receptorscapable of binding to the magnetic particles are present only in theserecesses.

By arranging the recesses on the surface of the magnetic sensor in thedetection space, the molecular receptors may be bound only to a specificarea of the surface of the magnetic sensor. By this structure, it ispossible to control an amount of bound magnetic particles in thedetection space and the like.

In further embodiments, it is characterized in that a first magneticfield generating means, which generates a magnetic field that keeps awaythe magnetic particles from the surface of the aforementioned magneticsensor so that the magnetic particles are not bound to the surfacethereof, is arranged in a position facing the surface.

By keeping the magnetic particles away from the surface of the magneticsensor, interference by unbound magnetic particles on the result ofdetection may be prevented. Also, by keeping unbound magnetic particlesaway from the surface of the magnetic sensor, operations such as washingout floating magnetic particles at the time of measurement and the likebecome unnecessary.

In further embodiments, it is characterized in that a second magneticfield generating means, which generates magnetic field that keeps themagnetic particles closer to the surface of the magnetic sensor, isfurther provided.

By this system, the binding of the magnetic particle to the surface ofthe magnetic sensor is expedited, and the measuring time may beshortened.

In further embodiments, it is characterized in that a second magneticfield generating means, which generates magnetic field that keeps themagnetic particles closer to the surface of the magnetic sensor, and amagnetic field device control means that operates the aforementionedfirst magnetic field generating means and the second magnetic fieldgenerating means alternately to generate magnetic field, so thatmagnetic particles not bound to the surface of said magnetic sensor arestirred, are still further provided.

By this system, the magnetic particles are stirred, and thus binding ofthe magnetic particles to the surface of the magnetic sensor isexpedited, and the measuring time may be shortened.

In further specific embodiments, in another biosensor of the presentinvention including: a magnetic sensor, in which a plurality of magneticfield detection elements are arranged, the elements each producing anoutput value corresponding to the intensity of the detected magneticfield; and a signal processing means that determines an amount ofmagnetic particles bound to said magnetic sensor based on respectiveoutput value obtained from said plurality of magnetic field detectionelements, the biosensor is characterized in that the signal processingmeans judges dispersion condition of the magnetic particles based on theoutput values obtained from the plurality of magnetic field detectionelements, after introducing the magnetic particles to the magneticsensor and before removing the unbound magnetic particles from themagnetic sensor.

By measuring in the weak magnetic field before unbound magneticparticles are removed by magnetic field, the amount of magneticparticles present on the surface of the magnetic sensor may be measuredregardless whether the magnetic particles are bound or unbound. By thisprocedure, it may be confirmed that the magnetic particles are dispersedon the surface of the magnetic sensor.

In a method of assaying an object using a biosensor of the presentinvention, in which the magnetic particles bind specifically to anobject that binds to the magnetic sensor, it is characterized in thatsaid the method of assaying an object includes: a step for determiningan amount of the magnetic particles bound to the magnetic sensor throughthe object using the biosensor; and a step for determining an amount ofthe aforementioned object based on the amount of the aforementionedmagnetic particles.

In another method of assaying an object of the present invention using abiosensor of the present invention, in which said magnetic particles arereplaceable with an object bound to said magnetic sensor reversibly, themethod is characterized in that this method includes: a step fordetermining an amount of the magnetic particles bound to the magneticsensor replacing the object using the biosensor; and a step fordetermining an amount of the object based on the amount of the magneticparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a cross section of a hall element and its vicinity;

FIG. 1B illustrates the change rate of magnetic flux density;

FIG. 2A illustrates the relationship of the output change rate of thehall elements in the weak and strong magnetic fields;

FIG. 2B illustrates the output change rate when any of the hall elementis not bound to the magnetic particle;

FIG. 2C illustrates the output change rate when the magnetic particle isbound to the surface of the sensor chip;

FIG. 3 illustrates a block diagram describing the biosensor circuit ofthe present embodiment;

FIG. 4 illustrates a schematic diagram showing a part of the biosensorof the present embodiment;

FIG. 5A illustrates a top view of the hall element 2,

FIG. 5B illustrates a sectional view at the dash-dotted line a;

FIG. 5C illustrates a sectional view at the dash-dotted line b;

FIG. 6 illustrates a diagram describing the selection method for hallelements in array form in the present embodiment;

FIG. 7 illustrates a schematic diagram of the whole biosensor of thefirst embodiment;

FIG. 8A illustrates a diagram describing the state of the magneticparticles on the surface of the sensor chip;

FIG. 8B illustrates a diagram describing the state of the magneticparticles on the surface of the sensor chip when magnetic field isgenerated from the upper coil;

FIG. 9 illustrates a flow chart describing the circuit action of theentire biosensor of the first embodiment;

FIG. 10 illustrates a frequency spectrum of the output signal of thehall element by Fourier transform;

FIG. 11 illustrates a flowchart describing the circuit action of theentire biosensor of the second embodiment;

FIG. 12 illustrates a diagram describing the result of the measurementin the example 1;

FIG. 13A illustrates a graph showing the result of the measurement inthe example 2 where the magnetic particles are unbound;

FIG. 13B illustrates a graph showing the result of the measurement inthe example 2 where the magnetic particles are bound;

FIG. 14 is a table showing the test result of the example 3; and

FIG. 15 illustrates a schematic diagram describing the solid phaseanalysis using conventional magnetic particles.

BEST MODE FOR CARRYING OUT THE INVENTION

The biosensor of the embodiment of the present invention will bedescribed by referring to diagrams as follows.

However, the invention is not limited to biosensors, but applicable tomagnetic sensors in general that measure an amount of magneticparticles.

First Embodiment

(System Configuration of Biosensor)

FIG. 3 shows the system configuration of the biosensor of the presentembodiment. The biosensor includes a sensor chip 1 to which a samplesolution is introduced to carry out measurement and a measurementequipment which has a magnetic field generator which applies a magneticfield to the sensor chip 1, and circuitry to communicate with the sensorchip 1.

The sensor chip 1 is integrated with an array of hall elements 9, anarray selection circuit 71, and an amplification circuit 81. Themeasurement equipment is installed with: a magnetic field generatorincluding an electromagnet 85, a power supplier for the electromagnet 86and a magnetic sensor for monitoring 87 which monitors the magneticfield by the electromagnet 85; a control circuit which controls sensorchip 1; a signal processing circuit (a signal processing means) whichprocesses the output signals from the hall elements; and other controlcircuits 82 (for example, a control circuit (a control means for themagnetic field device) for the power supplier of the magnetic fieldgenerator). The sensor chip 1 is changed to a new one in eachmeasurement.

(Configuration of the Sensor Chip)

FIG. 4 is a schematic diagram of a part of the sensor chip.

The sensor chip 1 is formed on a silicon substrate 11 by well-known CMOS(complementary metal-oxide semiconductor device) fabrication process. Onthe sensor chip 1, recesses 13 are formed spaced apart with apredetermined pitch. Under this recess 13, hall elements (magnetic fielddetection elements) are formed, and input and output of each hallelement are carried out through a gate electrode 30 and a metalinterconnect 4. The extreme surface is covered with silicon nitride filmor silicon oxide film prepared by the plasma CVD (chemical vapordeposition) method.

After fabricating hall elements, an array selection circuit 71 and anamplification circuit 81 on the silicon substrate 11 by the CMOSfabrication process, molecular receptors (antigen, antibody, DNA, RNAand the like), which bind magnetic particles to the surface of thesensor chip 1, may be immobilized on the sensor chip 1 by treating thesurface with silane coupling agent and the like.

The surface area of the hall element is supposed to be equal to themaximum cross section of the magnetic particle 51. In this way, thenumber of the magnetic particles 51 present in the magnetic fielddetectable by the hall element 2 may be kept about 1. Therefore, whenthe measurement is carried out by detecting the presence or absence ofone magnetic particle 51 by the hall element 2, the detection of two ormore of the magnetic particles 51 by the hall element may be prevented,and accurate measurements may be carried out. However, the measurementof the present invention is not limited to detecting the presence orabsence of one magnetic particle 51 by the hall element. That is, thesurface area of the hall element 2 may be equal to the maximum crosssection of a plurality of the magnetic particles and a plurality ofmagnetic particles may be detected by the hall element.

Further, the arrangement of the hall elements, the space between therecesses 13 and the like are not limited in particular. As shown in theprinciple of detection of FIGS. 1A and 1B, the change rate of themagnetic flux density is changed not only at the hall element 2 a wherethe magnetic particle is directly on the top, but also in the regionnear the magnetic particle such as at the hall element 2 b where themagnetic flux density is reduced. Therefore, the molecular receptors 61may be immobilized on the whole surface of the sensor chip 1 to detectthe change in the magnetic flux density near the magnetic particle.Further, since the change rate in the area directly under the magneticparticle 51 where the magnetic flux density is increased is greater thanin the surrounding area where the magnetic flux density is decreased asshown in FIG. 1B, the molecular receptors may be selectively immobilizeddirectly on top of the hall elements so that the magnetic particles arebound only directly on top of the hall elements. Still further, hallelements may be arranged by separating with a distance of, for example,the hall element 2 a and 2 c as shown in FIG. 1A, so that the magneticparticle bound on top of the neighboring hall element has no influence.

(Structure of Hall Elements)

Next, the structure of hall elements will be described.

FIG. 5A is a top view of the hall element 2, FIG. 5B is a sectional viewat the dash-dotted line a, and FIG. 5C is a sectional view at thedash-dotted line b. This hall element 2 is composed of a gate electrode30, a source electrode 31, a drain electrode 32, an output electrode 33,34 and an insulation layer 35, and formed in a P well region 36. Theconfiguration is the same as then type MOSFET except the outputelectrodes, and the metal interconnects to each electrode are omitted inthe figures. The output electrodes 33, 34 are arranged so that thecurrent flows vertically to the magnetic flux, which is formed almostvertically to the surface of the sensor chip, and the current flowingbetween the source drain electrodes.

The operation of this hall element 2 will be described. A bias isapplied to the gate electrode 30, source electrode 31 and drainelectrode 32 and the operation condition is similar to MOSFET. It ispreferable that the operation condition at this time is in a linearregion. If there is no external magnetic flux in this condition, the twooutput electrodes 33, 34 are in an equal potential. When an externalmagnetic flux is applied vertically to the surface of the hall element,a voltage proportional to the magnetic flux density is generated betweenthe output electrodes 33 and 34 as a differential voltage.

(Array Arrangement of Hall Elements and Selection Method of Each HallElement)

Next, the arrangement of the hall elements on the sensor chip and themethod for selecting each hall element and obtaining outputs will bedescribed.

A source electrode, a drain electrode and a pair of output electrodes ofeach hall element (E(0,0), E(0,1), . . . ) are connected to V_(L),V_(H), OUT1, OUT2 through switches (R0, R1 . . . ) and the hall elementsin the same row are connected in common. Also, the gate electrodes inthe same column are connected to a common gate electrode interconnectsC0, C1, . . . . In FIG. 6 columns and rows are shown in X and Y,respectively. V_(L), V_(H) are the interconnects to supply bias to thehall element, and OUT1, OUT2 are interconnects to output from the hallelements to the amplification circuit.

The case of selecting the hall element E(0,0) will be described. Onlythe switch R0 is set on and switches R1, R2, . . . are set off. Further,only the gate electrode interconnect C0 is set at a voltage that thehall element is in the active condition but the gate electrodeinterconnects C1, C2, . . . are set at the voltage that the hallelectrodes are not in the active condition, that is there is no currentflowing between the source and drain even if a bias is applied to thesource electrode and drain electrode.

At this time, V_(L), V_(H) are applied to the hall element E(0,0) andthe other elements in the same row and the source electrode and drainelectrode, but current only flows through the hall element E(0,0). Thevoltage corresponding to the magnetic flux density is generated at theoutput electrodes of the hall element E(0,0). Since the output electrodeof other hall elements in the same column are not in the activecondition, the output voltage of the hall element E(0,0) is outputted toOUT1 and OUT2 as it is. In this configuration, even if the number ofarrays is increased, the number of interconnects in the array is thesame and only switches are added at the edge. Therefore, the area of asensor chip is almost proportional to the number of arrays, and a sensorchip with a large number of hall elements may be composed easily.

(Configuration of Electromagnet)

FIG. 7 shows the arrangement of an electromagnet. In the presentembodiment, the electromagnet 85 includes an upper coil 24 that isarranged on the opposite location of the surface of the sensor chip 1and a lower coil 25 that is arranged on the reverse side of the sensorchip 1.

The upper coil 24 is a means for applying external magnetic field andgenerates a magnetic field with a magnetic flux vertical to the sensorchip 1. This magnetic field is detected by the hall elements, and in thepresent embodiment, a weak magnetic field with a magnetic permeabilityof the bound magnetic particles falling with in a range from initialmagnetic permeability to maximum magnetic permeability and a strongmagnetic field with magnetization that at least some of the magneticparticles becomes saturated are formed on the surfaces of the sensorchip. Further, in the present embodiment, this weak magnetic field is anAC magnetic field and the strong magnetic field is formed by adding astrong DC magnetic field to this AC magnetic field.

Still further, the upper coil 24 functions as a first magnetic fieldgenerating means which keeps magnetic particles away from the surface ofthe sensor chip 1 and is turned on when the magnetic particles areintroduced to the sensor chip 1 and generates a magnetic field in whichthe magnetic flux density is increased in proportion to the distancefrom the surface of the sensor chip 1. By the operation of this uppercoil 24, floating magnetic particles which are not bound to the surfaceof the sensor chip 1 are kept away from the surface of the sensor chip1, and thus unbound magnetic particles do not affect the magnetic fluxthat is detected by the hall elements.

Furthermore, in place of the coil, for example, a permanent magnet maybe used.

The lower coil 25 functions as a second magnetic field generating meanswhich keeps magnetic particles closer to the surface of the sensor chip1 and forms a magnetic field which is not for detecting magneticparticles. When magnetic particles are introduced to the sensor chip 1,the lower coil 25 forms a magnetic field in which the magnetic fluxdensity is increased with getting nearer to the surface of the sensorchip 1. By the operation of this lower coil 25, magnetic particles areattracted to the surface of the sensor chip 1, and the time from theintroduction of the magnetic particles to their binding to the surfaceof the sensor chip 1 is shortened. It is effective for magneticparticles with diameter of 1 μm or less in particular because they havea tendency not to go down with gravity alone.

Also, in place of the coil, for example, a permanent magnet may be used.

Further, by operating the upper coil 24 and the lower coil 25alternately by the magnetic device control means, the magnetic particlesmove up and down, and the binding between the object and magneticparticles may be promoted.

FIGS. 8A and 8B show the views of the surface of the sensor chip 1 whenthe magnetic field is formed by the upper coil 24 with the introductionof the magnetic particles. The floating unbound magnetic particles areattracted upward, but some of the floating magnetic particles near thesurface of the sensor chip 1 and the one bound to the surface of thesensor chip 1 are magnetized by the magnetic field by the coil and areattracted each other. As shown in FIG. 8A, when the magnetic field isnot applied, the magnetic particles 51 is not magnetized and some arebound to the surface of the sensor chip 1 through the object 22, someare present on the surface of the sensor chip 1 without binding and someare floating. When the external magnetic field is applied to this, themagnetic particles 51 are magnetized and attracted each other by thismagnetization as shown in FIG. 8B. And to the magnetic particle 51 boundto the surface of sensor chip 1, other magnetic particles 51 are linkedin a column along the direction of the magnetic flux of the externalmagnetic field. This condition makes the change in the magnetic flux atthe hall element 2 a greater than the condition of the presence ofsingle magnetic particle 51 does. Also, the magnetization of themagnetic particles 51 has a tendency to be saturated more easily, makingthe measurement with a higher sensitivity possible.

(Operation of the Biosensor)

Next, the circuit operation of the biosensor of the present inventionshown in a flow chart of FIG. 9 will be described.

At step S101, a magnetic field is generated by applying the current tothe lower coil 25 with the magnetic particles placed over the sensorchip 1 and the magnetic particles 51 are attracted to the surface of thesensor chip. At this time the current applied to the lower coil 25 maybe direct current or alternating current. Further, the current appliedto the lower coil 25 is controlled so that the predetermined magneticfield is achieved, by measuring the magnetic field generated by thelower coil 25 using the magnetic sensor for monitoring 87.

At step S102, the magnetic field by the lower coil 25 is turned off.

At step S103, the current is applied to the upper coil 24 to generate amagnetic field to keep the magnetic particles 51 away from the surfaceof the sensor chip 1. At this time the current applied to the upper coil24 may be direct current or alternating current. Further, the currentapplied to the upper coil 24 is controlled so that the predeterminedmagnetic field is achieved, by measuring the magnetic field generated bythe upper coil 24 using the magnetic sensor for monitoring 87.

At step S104, the magnetic field by the upper coil 24 is turned off.

At step S105, the magnetic particles 51 is stirred in a sample solution,until the predetermined time or number is reached when the binding ofthe magnetic particles 51 to the surface of the sensor chip 1 iscompleted, by proceeding into step S101 again or step 105 and repeatingsteps of S101-S104.

At step S106, the current is applied to the upper coil 24 to generate amagnetic field to keep the magnetic particles 51 not bound to thesurface of the sensor chip 1 away from the surface of the sensor chip 1,making possible to detect only the magnetic particles 51 bound to thesensor chip 1.

Step S107 is a waiting period until the predetermined time forcompleting the removal of the unbound magnetic particles 51 from thesurface of the sensor chip 1.

After completing the removal of the unbound magnetic particles, in stepS108, the strong magnetic field is generated from the upper coil 24 toobtain the output signals from the hall elements. Next, in step S109,the weak magnetic field is generated from the upper coil 24 to obtainthe output signals from the hall elements. In particular, an addresssignal for selecting a specific hall element is sent from the sensorchip control circuit 82 in the measurement equipment to the arrayselection circuit 71 in the sensor chip 1. The array selection circuit71 selects the designated hall element based on this address signal asdescribed above. The output signal from the hall element is amplified bythe amplification circuit 81 on the sensor chip. The amplified outputsignal is stored in the memory 83.

At step S110, it is decided whether the signals have been obtained ornot from all the hall elements from which the output signal should beobtained, and if not, step S108 is repeated. By these procedures outputsignals from all the hall elements are obtained.

At step S111, the magnetic field by the upper coil 24 is turned off.

At step S112, the output values of each hall element at the strong andweak magnetic fields obtained at steps S108 and S109 are retrieved fromthe memory 83, and the number of bound magnetic particles is determinedby comparing the output values of the hall elements in the signalprocessing circuit 82 .

(Determination of Number of Magnetic Particles)

Next, described will be the comparison of the output values anddetermination of the number of magnetic particles by the signalprocessing circuit 82, after obtaining the output values of the hallelements as described above.

In an output value of an arbitrary hall element, the output change ratesof this hall element to a small change in the AC magnetic fieldcomponent of the upper coil 24 are calculated in the cases of the strongmagnetic field and the weak magnetic field, and it is judged whether therespective output change rate is different or not. That is, when themagnetic particles 51 are not bound directly on the top or near a hallelement as described above, the output change rates of this hall elementin the strong and the weak magnetic fields are the same, but when themagnetic particles are bound directly on the top or near the hallelement, the ratio of the output value of this hall element to theintensity of the magnetic field of the upper coil is different in thestrong and the weak magnetic fields. Therefore, if the judgment is madethat the output change rates are the same, then it is judged that themagnetic particles 51 are not bound directly on top or near the hallelement, and if the output change rates are different, then it is judgedthat the magnetic particles 51 are bound directly on top or near thehall element. Further, in accordance with the size of the differentialof the output change rate and the sign of the output change rate, it maybe judged that the magnetic particles are not bound directly on top butnear the hall element, and the like.

This judgment action is repeated in every hall element, and based onthese judgment the number of bound magnetic particles is determined.

(Method for Measurement of Object Using the Biosensor)

By using the biosensor described above, and measuring the number ofmagnetic particles bound to the sensor chip, the concentration and thelike of an object in a sample solution can be measured.

In the example of FIGS. 1A and 1B, antibodies are fixed on the surfaceof the sensor chip 1 as molecular receptors 61 that bind specifically tothe object 62. Further, the magnetic particle 51 includes on the surfacesecondary molecular receptors 63 and this secondary molecular receptors63 binds specifically with the object 62. Thus, the magnetic particle 51specifically binds to the object 22 that is bound to the surface of thesensor chip 1 through the molecular receptor 61. By this procedure, theamount of the object 62 can be determined based on the measurement ofthe amount of the magnetic particles bound to the sensor chip 1 throughthe object 22, the molecular receptor 61 and the like.

The method of measuring of the objects using the biosensor is notlimited to this method, and for example, the molecule that bind to thesurface of the sensor chip 1 competitively with the object may be themagnetic particles. In this case, the amount of the magnetic particleswhich bind replacing the object is determined using the biosensor, andthe amount of the competitive object can be determined based on theamount of the magnetic particles.

Second Embodiment

Next, the second embodiment of the present invention will be described.

The biosensor of the second embodiment is composed almost the same wayas the biosensor of the first embodiment, but there is a difference inthe manner of comparison of the output values and determination of thenumber of the magnetic particles by the signal processing circuit 82after obtaining the output values from the hall elements. These aredescribed below.

In this embodiment, first the dispersion of the output valuedistribution in the weak magnetic field is calculated from the outputvalues of all hall elements when the weak magnetic field is applied.Next, the dispersion of the output value distribution in the strongmagnetic field is calculated from the output values of all hall elementswhen the strong magnetic field is applied. Then, the differentialbetween the dispersions of the output value distribution in the weakmagnetic field and in the strong magnetic field is obtained, and theamount of the magnetic particles bound to the sensor chip is determinedbased on this differential.

That is, the variation of the output values from a plurality of magneticfield detection element, which is caused by the magnetic particles boundto the magnetic sensor, is converted to the dispersion, and the amountof bound magnetic particles is determined based on the degree of thisvariation.

Also, as described above, the change rate of the magnetic flux densityat the hall element to the magnetic field by the coil is positivedirectly under the magnetic particle 51 and negative at the location alittle away from the directly under as shown in FIG. 1B. When themagnetic particles are not present near the hall element, the changerate of the magnetic flux density is 0. Regardless of the change ratebeing positive or negative, the amount of the change when the magneticpermeability of the magnetic particles 51 is between the initialpermeability and the maximum permeability is greater than the changerate when a part or the whole magnetization of the magnetic particle 51is saturated. Thus, the differentials of the dispersions of the outputvalues of all the hall elements in the weak magnetic field and in thestrong magnetic field are proportional to the amount of the magneticparticles bound to the sensor chip, wherein the magnetic particles 51 isbetween the initial permeability and the maximum permeability in theweak magnetic field, and the magnetization of a part or whole of themagnetic particles are saturated in the strong magnetic field.

Further, in the ideal condition where the sensitivity of all the hallelements is the same, the deviation of the output values of all the hallelements is 0 when the magnetic particles are not bound in the weakmagnetic region. When the magnetic particles 51 are bound on top of somehall elements, the dispersion is dependent on the amount of the boundmagnetic particles because the output values of the hall elements thatare bound to the magnetic particles 51 are changed. Therefore, theamount of bound magnetic particles 51 can be obtained. However, inreality, the sensitivity of the hall elements on the sensor chip 1 mayvary to some extent due to the production process and the deviation isnot 0 when the magnetic particles 51 are not bound. However, each of thedispersion of the output value distribution in the strong magnetic fieldand in the weak magnetic field is equal, and the differential betweenthe dispersions is 0. When magnetic particles are bound on top of someof the hall elements, the change rate may be plus or minus depending onthe location of the binding but it is greater in bound condition than inthe state where the magnetic particles 51 is not bound. Further, sincethe amount of change in the change rate is greater in the weak magneticfield than in the strong magnetic field, the dispersion is also greaterin the weak magnetic field than in the strong magnetic field when themagnetic particles 51 are bound. Thus, in the condition where themagnetic particles 51 are bound, there is a differential of thedispersions of the output vales of all the hall elements in the weakmagnetic field and in the strong magnetic field, wherein in the weakmagnetic field, the magnetic permeability of the magnetic particle 51falls between the initial permeability and in the strong magnetic field,the maximum permeability and the magnetization of some or all of themagnetic particles are saturated.

In an example of real measurements, as shown in FIG. 2B, when themagnetic particles are not bound, the output change rates aredistributed around a line with a slope of 1, confirming that thedispersions in the weak magnetic field and in the strong magnetic fieldare almost the same.

When the magnetic particles are in bound condition, the output changerate in the strong magnetic field in the weak magnetic field is greaterand the dispersion of the plots is spreading to the direction of thevertical axis as clearly seen in FIG. 2C. That is, the dispersion in theweak magnetic field becomes greater than that in the strong magneticfield and it is confirmed that the binding of the magnetic particles canbe detected by obtaining the differential.

Third Embodiment

Next, the third embodiment of the present invention will be described.The biosensor of the third embodiment is composed in almost the same wayas the biosensor in the first embodiment but the configuration of thesignal processing circuit 82 is a little different. Following is thedescription.

In the present embodiment, the signal processing circuit 82 furtherincludes: a noise component prediction part that predicts the noisecomponent from the frequency component other than that corresponds tothe AC magnetic field in the output value of hall elements; and a noisecomponent removal part that removes, based on the predicted noisecomponent, the noise component from the frequency component whichcorresponds to the AC magnetic field in the output value of hallelements.

The noise component prediction part is composed of an AD converter and ahigh speed Fourier converter, and carries out Fourier conversion of theoutput signal of the hall element and calculates the predicted noiselevel in the frequency component that corresponds to the AC magneticfield, based on the trend of the output level of the frequency spectraother than the frequency corresponding to the AC magnetic field.

The frequency spectra obtained by Fourier transformation of the outputsignal of the hall element is shown in FIG. 10. The alternating currentcomponent of the magnetic flux on the sensor chip 1, by the externalmagnetic field applied to the sensor chip 1 and the magnetic particle51, is composed of the frequency of the alternating current component ofthe external magnetic field and the frequency component corresponding toits harmonic waves. Since the output signals of the sensor containsnoise, the Fourier conversion results in the spectra, as shown in thefigure, covering the entire range of frequencies. As shown in the figurethe signal component appears at the specific frequencies. The noisecomponent includes mainly thermal noise that is not dependent onfrequencies and the flicker noise that is proportional to the reciprocalof frequency. Since these noise components change against frequenciesalmost continuously, the noise level of the frequency, at which thesignal appears, can be estimated from the spectra of the neighboringfrequencies. Thus, the true signal component can be obtained bysubtracting this noise level from the level of the sensor output.

The noise removal part calculates: a ratio of the true signal component,after removing the noise level based on the calculated noise level bythe noise component prediction part, to the entire frequency componentcorresponding to the AC magnetic field; and obtains the true signalcomponent by obtaining the output corresponding the ratio, when thefrequency component corresponding to the AC magnetic field is extractedfrom the output signal of the hall element.

By doing this, the noise shown in FIG. 2A that varies in all thedirections may be reduced, and the accuracy of the measurement becomesexcellent.

Fourth Embodiment

Next, the fourth embodiment of the present invention will be described.

The biosensor of the fourth embodiment is composed similar to thebiosensor of the first embodiment but the action in the measuring isdifferent. Following is the description.

The action of the biosensor of the present embodiment is shown in a flowchart of FIG. 11. The description will not be made on the same point ofthe first embodiment.

At step S201, the magnetic particles 51 are introduced to the sensorchip 1 and stand until the predetermined binding of the magneticparticles 51 to the sensor chip 1 is completed. At this point, thebinding of the magnetic particles 51 to the sensor chip 1 is promoted bystirring the sample solution by activating the upper coil 24 and lowercoil 25 alternately as in the steps S101-S105 in the first embodiment.

At step S202, the weak magnetic field is generated by the upper coil 24,and at step S203, the output signal of a hall element is obtained andstored in the memory 83. These procedures are repeated until the outputsignals from the predetermined number of the hall elements are obtained(step S204).

At step S205, the predetermined magnetic field is generated by the uppercoil 24, and stand for the predetermined time for removing of theunbound magnetic particles 51 from the surface of the sensor chip 1 iscompleted.

At step S206, the weak magnetic field is generated from the upper coil24 and the output signal of a hall element is obtained and stored in thememory 83. At step S207, it is judged whether the signals are retrievedfrom all the hall elements, from which the signals should be retrieved,and in the case the signal not retrieved (No.) then S206 is repeated. Bythese procedures, the output signals of all the hall elements areobtained.

At step S208, the strong magnetic field is generated by the coil 24, thesignal of a hall element is obtained and stored in the memory 83. Atstep S209, it is judged whether the signals are retrieved from all thehall elements, from which the signals should be retrieved, and in thecase the signal not retrieved (No.) then S208 is repeated. By theseprocedures, the output signals of all the hall elements are obtained.When all the signals are obtained, the magnetic field of the upper coil24 is shut off (step S210).

At step S211, the output value of each hall element obtained at stepS203 and S208 is retrieved from the memory 83, and the output values ofthe hall elements are compared in the signal processing circuit 82.Then, the number of magnetic particles present on the sensor chip 1,regardless of whether bound or unbound, is determined before the unboundmagnetic particles are removed from the sensor chip 1. By doing this, itcan be confirmed whether the measurement is carried out under the statewhere the magnetic particles are evenly disperse.

At step S212, the output values of hall elements obtained at step S206and S208 are retrieved from the memory 83 and compared in the signalprocessing circuit 82, and the number of bound magnetic particles isdetermined.

The method for determining the number of magnetic particles shown in thefirst and the second embodiment may be used as a method for determiningthe number of magnetic particles in the steps S211 and S212.

By carrying out the measuring in the strong magnetic field after themeasuring in the weak magnetic field, some of the magnetic particlesbound to the surface of the magnetic sensor are released and the signalsmay be obtained in the closer condition where the magnetic particles arenot bound, and thus more accurate measurement can be made.

The invention described above will be described based on the examples.

EXAMPLE 1

The hall elements shaped as shown in FIGS. 5A to 5C that were arrangedin array like arrangement and further, an array selection circuit and anamplification circuit were fabricated on a same silicon substrate. Thedistance between the source electrode 31 and the drain electrode 32 wasabout 6.4 μm and the distance between the sensitive face, that was achannel formed under the gate electrode 30 and the surface of theinsulation layer 12 is about 2.8 μm. The pitch of the hall elementsarranged array-like was 12.8 μm. The voltage applied between the sourceelectrode of the hall element selected by the array selection circuitand the drain electrode was about 4V, and between the source electrodeand the gate electrode was about 5V.

At the time of the measurement, an AC magnetic field with an effectiveintensity of 50 gauss in the weak magnetic field was applied, and in thecase of the strong magnetic field, in addition to the AC magnetic fieldwith an effective intensity of 50 gauss, the DC magnetic field with aintensity of 200 gauss was applied by a coil.

On the sensor chip, magnetic particles with a diameter of 4.5 μm made byDynal Inc. (Commercial Name: DYNABEADS) were bound. The result of themeasurement is shown in FIG. 12. The ratio of the amount of the changeof the alternating current component of the output of each hall elementto the small change in the component of the AC magnetic field applied bythe coil is shown. The measurements were carried out on the 128 hallelements on the sensor chip, and the figure shows the result of somehall elements. In the weak and strong magnetic fields, there was almostno difference in the outputs of the hall elements not bound to themagnetic particles against the magnetic field by the coil. However, theoutputs of the 13^(th) and 15^(th) hall elements that were bound to themagnetic particles were greater in the weak magnetic field than in thestrong magnetic field.

If the magnetization of the magnetic particles was completely saturatedin the strong magnetic field, the output of the sensor not bound to themagnetic particles should have been the same level, but, since themagnetization was not saturated, there was a difference even in thestrong magnetic field. However, where the magnetic particles were notbound, there was no difference between the weak and strong magneticfield, and where the magnetic particles were bound, a clear differencewas seen between the strong and weak magnetic field; and thus themagnetic particles bound to the surface can be detected (the method ofdetermining the number of magnetic particles shown in the firstembodiment).

Since the magnetic particles with a size similar to the hall elementswere used in FIG. 12, the bound magnetic particles can be clearlydetected. Further, the average deviations of all the 128 hall elementsin the strong and weak magnetic fields are 0.56% and 1.48%,respectively, and from this differential the presence or absence of themagnetic particles can be judged (the method of determining the numberof magnetic particles shown in the second embodiment).

EXAMPLE 2

FIG. 13A shows the result of measurement when the magnetic particleswere not bound to the sensor chip that was similar to the one in theexample 1, and FIG. 13B shows the result of the measurement when thesensor chip was bound to the magnetic particles with a diameter of 1 μmmade by Dynal Inc. The vertical axis represents the ratio of the amountof the change in the alternating current component of the output of eachhall element to the micro-change in the AC magnetic field componentapplied by the coil. The measurements were carried out in each of allthe 128 hall elements, and the figure shows the results of only some ofthe hall elements. In this case the magnetic particles were smallcompared to the size of the hall element, and thus a plurality ofmagnetic particles could bind to one hall element. However, since thevolume was also small and the magnetization by the magnetic fieldapplied by the coil was small, the change in the magnetic flux densityon the hall element was small.

FIG. 13A shows that the output value of each hall element is varyingalthough the magnetic particles are not bound. On the other hand in FIG.13B where the magnetic particles are bound, it can not be judged whetherthe magnetic particles are bound or not, because the magnetization ofthe magnetic particles is small and the output value from each hallelement is varied.

The differential of the average deviations of the output of all the 128elements between the strong and weak magnetic fields were 0.02% withoutmagnetic particles and 0.12% with bound magnetic particles. This resultindicates that even if the judgment can not be made on the binding ofthe magnetic particles from the outputs of each hall element due to themagnetization of the magnetic particles being too small, the judgmentstill can be made on the binding of the magnetic particles by obtainingthe differential of the average deviations of the outputs of a pluralityof hall elements (the method of determining the number of magneticparticles shown in the second embodiment).

EXAMPLE 3

The magnetic particles with a diameter of 1 μm made by Dynal Inc. werebound to the sensor chip on which the 256 hall elements were arranged.At this time, an antigen derived from Haemophilus influenzae was used asan object. FIG. 14 shows the average deviation of the outputs of thehall elements against the antigen concentration in the weak and strongmagnetic fields, and the differential of the average deviation. As seenin the figure, the differential of the average deviation increases withthe increase of the antigen concentration.

This result indicates that even if the judgment can not be made on thebinding of the magnetic particles from the outputs of each hall elementdue to the magnetization of the magnetic particles being too small, thejudgment still can be made on the binding of the magnetic particles byobtaining the differential of the average deviations of the outputs of aplurality of hall elements. Further, it was confirmed that even if theantigen concentration is 1 ng/ml or lower, the measurement of theantigen concentration was possible based on the number of bound magneticparticles.

INDUSTRIAL APPLICABILITY

The biosensor of the present invention can be used for measuring theamount of magnetic particles, and further, for clinicaldiagnosis/detection, analysis of the genes and the like, byimmunological means that detects antigen, antibody, DNA(Deoxyribonucleic Acid), RNA (Ribonucleic Acid) and the like, using thespecific binding between certain molecules, such as the binding of anantigen labeled with magnetic particles to the antibody to this antigenand the like.

Since the biosensor of the present invention determines the amount ofthe magnetic particles bound to the magnetic sensor, based on thedispersion of the distribution of the output values of a plurality ofthe magnetic field detection elements, or based on the output valuechange in the magnetic field detection elements against the change inintensity of the external magnetic field, it is possible to makeaccurate measurements. Further, since the external magnetic fields ofgreatly different intensities are applied in the condition where themagnetic particles are bound, and the measurements are carried out basedon the output values of the magnetic field elements in each magneticfield, even if the characteristics such as sensitivity and the like arevaried, a refernce value of the magnetic field may be obtained by eachindividual magnetic field detection element and also the reference valuemay be obtained in the condition where the magnetic particles andobjects are introduced, it is possible to make measurements rapidly andaccurately. Further, using hall elements as the magnetic field detectionelements for this purpose, the accuracy of measurement becomesexcellent. Still further, the biosensor becomes inexpensive and compactby using hall elements as the magnetic field detection elements.

Still further, since the method of measuring objects in the presentinvention uses the biosensor described above, it is possible to carryout the measurement rapidly, simply and accurately without washing-outthe unbound labeled material.

1. A sensor comprising a magnetic sensor, which has a plurality ofmagnetic field detection elements arranged two dimensionally in X rowsand Y columns (X and Y are natural numbers), said detection elementseach generating an output value according to the intensity of a detectedmagnetic field, and measures an amount of magnetic particles on saidmagnetic sensor based on said output values, wherein said sensor has asignal processing means by which said amount of magnetic particles isdetermined based on a dispersion of an output value distributionobtained from said output values of the plurality of magnetic fielddetection elements.
 2. A sensor according to claim 1, wherein saidmagnetic particles bind to an object that is bound to said magneticsensor, and said signal processing means determines the amount ofmagnetic particles bound to said magnetic sensor through said object andfurther an amount of said object based on the amount of said magneticparticles.
 3. A biosensor according to claim 2, wherein said signalprocessing means determines the amount of said bound magnetic particlesbased on the difference between the dispersion of said output valuedistribution and the dispersion of the reference distribution, thelatter distribution being obtained from the output value of theplurality of magnetic field detection elements in the state where themagnetic particles are not bound to said magnetic sensor.
 4. A biosensoraccording to claim 2, comprising a means for applying external magneticfields of greatly different intensities to said magnetic sensor bound tosaid magnetic particles, wherein one of said external magnetic fields ofgreatly different intensities is such a strong magnetic field that themagnetization intensity of at least some of the bound magnetic particlesbecomes saturated, and another external magnetic field is such a weakmagnetic field that said bound magnetic particles each have a magneticpermeability falling within a range from an initial magneticpermeability to a maximum magnetic permeability, and wherein said signalprocessing means determines an amount of said bound magnetic particlesbased on the difference between the dispersion of said output valuedistribution when said strong magnetic field is applied and thedispersion of said output value distribution when said weak magneticfield is applied.
 5. A biosensor comprising a magnetic sensor, which hasa plurality of magnetic field detection elements arranged twodimensionally in X rows and Y columns (X and Y are natural numbers),said detection elements each generating an output value according to theintensity of a detected magnetic field, and measures an amount ofmagnetic particles on said magnetic sensor based on said output values,further comprising: a means for applying external magnetic field ofgreatly different intensities to said magnetic sensor bound to saidmagnetic particles; a signal processing means that determines the amountof said bound magnetic particles by comparing output values ofrespective said magnetic field detection elements when the externalmagnetic field of greatly different intensities is applied, and whereinone of said external magnetic field of greatly different intensities issuch a strong magnetic field that the magnetization intensity of atleast some of said bound magnetic particles becomes saturated, andanother external magnetic field is such a weak magnetic field that ofsaid bound magnetic particles each have the magnetic permeabilityfalling within a range from an initial magnetic permeability to amaximum magnetic permeability.
 6. A biosensor according to claim 5,wherein said strong magnetic field changes in intensity within such arange that at least some of said bound magnetic particles becomessaturated, and said weak magnetic field changes in intensity within sucha range that the permeability of said magnetic particles changes fromthe initial magnetic permeability to the maximum magnetic permeability,and said signal processing means obtains changes in output values ofsaid magnetic field detection elements according to changes in intensityof the external magnetic field, when external magnetic fields of greatlydifferent intensities including said strong magnetic field and said weakmagnetic field are applied, and determines the amount of said boundmagnetic particles based on differentials of the changes in these outputvalues.
 7. A biosensor according to claim 4, wherein the output valuesare obtained from said plurality of said magnetic field detectionelements first by applying said weak magnetic field by said means forapplying an external magnetic field, and then another set of outputvalues is obtained from said plurality of said magnetic field detectionelements by applying said strong magnetic field by said means forapplying an external magnetic field.
 8. A biosensor according to claim4, wherein said means for applying an external magnetic field applies amagnetic field vertically to said magnetic sensor.
 9. A biosensoraccording to 4, wherein said means for applying an external magneticfield applies a DC magnetic field.
 10. A biosensor according to claim 4,wherein said means for applying an external magnetic field applies an ACmagnetic field.
 11. A biosensor according to claim 4, wherein said weakmagnetic field is an AC magnetic field with such an intensity that themagnetic permeability of said each bound magnetic particle falls withina range from the initial magnetic permeability to the maximum magneticpermeability, and said strong magnetic field is an external magneticfield of said AC magnetic field plus a DC magnetic field, with such anintensity that the magnetization intensity of at least some of saidbound magnetic particles becomes saturated.
 12. A biosensor according toclaim 10, wherein said signal processing means further includes: a noiseprediction means that predicts noise components from frequencycomponents other than those corresponding to said AC magnetic field,said noise components being included in output values of said magneticfield detection elements; and a noise removal means that removes noisecomponents from frequency components corresponding to said AC magneticfield included in output values from said magnetic field detectionelements based on the noise components predicted by said noiseprediction means.
 13. A biosensor according to claim 4, wherein themagnetic particles bound to said magnetic sensor are associated withother magnetic particles in a direction of the magnetic flux formed bysaid external magnetic field.
 14. A biosensor according to claim 2,wherein said magnetic field detection elements generate output values inproportion to a magnetic flux density of a magnetic flux formed in adetection space where a magnetic field may be detected.
 15. A biosensoraccording to claim 2, wherein said magnetic field detection elementseach comprise a hall element.
 16. A biosensor according to claim 15,comprising further a selection means for selecting an arbitrary elementamong said plurality of said magnetic field detection elements andobtaining an output value therefrom.
 17. A biosensor according to claim16, comprising still further a signal amplification circuit thatamplifies the output value of said magnetic field detection elementselected by said selection means, wherein said magnetic sensor, saidselection means and the signal amplification circuit are formed on achip.
 18. A biosensor according to claim 15, wherein said hall elementcomprises: a pair of current terminals; a gate electrode that controls acurrent flowing between said pair of current terminals; and a pair ofoutput terminals that are disposed so that a current flows in almostvertical direction against the current flowing between said pair ofcurrent terminals.
 19. A biosensor according to claim 18 including: saidgate electrode that is connected to a gate electrode interconnect whichis common for said hall elements arranged in a same row; said pair ofcurrent terminals that are connected to a pair of current terminalinterconnects which are common for said hall elements arranged in a samecolumn; and said pair of output terminals that are connected to a pairof output terminals which are common for said hall elements arranged ina same column, and wherein said selection means selects an arbitraryelement from a plurality of hall elements and obtains output valuethereof, by selecting one from Y numbers of gate electrodeinterconnects, a pair from X numbers of pairs of current terminalinterconnects and a pair from X numbers of pairs of output terminalinterconnects.
 20. A biosensor according to claim 2, wherein, in eachdetection space where each magnetic field may be detected by saidmagnetic field detection elements, an area of a vertical cross sectionof a magnetic flux formed on a surface of said magnetic sensor is almostthe same as a maximum cross section area of said magnetic particle. 21.A biosensor according to claim 2, wherein said each magnetic fielddetection element is arranged with a space so that each element detectsdifferent magnetic particles from each other.
 22. A biosensor accordingto claim 2, wherein a surface of said magnetic sensor is treated so thatmolecular receptors capable of binding to said magnetic particles may beimmobilized thereto.
 23. A biosensor according to claim 22, wherein thesurface of said magnetic sensor is treated so that the molecularreceptors capable of binding to said magnetic particles may beimmobilized selectively to a specific area.
 24. A biosensor according toclaim 2, in which recesses corresponding to the magnetic particles insize are formed on the surface of said magnetic sensor in a detectionspace capable of magnetic field detection, wherein the molecularreceptors capable of binding to magnetic particles are present only inthese recesses.
 25. A biosensor according to claim 2, wherein a firstmagnetic field generating means, which generates a magnetic field thatkeeps away the magnetic particles from the surface of said magneticsensor so that the magnetic particles are not bound to the surfacethereof, is provided facing said surface.
 26. A biosensor according toclaim 2, wherein a second magnetic field generating means, whichgenerates a magnetic field that keeps said magnetic particles closer tothe surface of said magnetic sensor, is further provided.
 27. Abiosensor according to claim 25, further comprising: a second magneticfield generating means, which generates a magnetic field that keeps saidmagnetic particles closer to the surface of said magnetic sensor; and amagnetic field device control means that operates said first magneticfield generating means and said second magnetic field generating meansalternately, to generate a magnetic field so that magnetic particles notbound to the surface of said magnetic sensor are stirred.
 28. Abiosensor comprising a magnetic sensor, in which a plurality of magneticfield detection elements are arranged, said elements each producing anoutput value corresponding to the intensity of the detected magneticfield; and a signal processing means that determines an amount ofmagnetic each particles bound to said magnetic sensor based onrespective output values obtained from said plurality of magnetic fielddetection elements, wherein said signal processing means judges thedispersion condition of said magnetic particles based on the outputvalues obtained from said plurality of magnetic field detectionelements, after introducing said magnetic particles to said magneticsensor and before removing said unbound magnetic particles from saidmagnetic sensor.
 29. A method of assaying an object using a biosensoraccording to claim 2, in which said magnetic particles bind specificallyto an object that binds to said magnetic sensor, wherein said methodcomprises the steps of: determining an amount of said magnetic particlesbound to said magnetic sensor through said object using said biosensor;and determining an amount of said object based on the amount of saidmagnetic particles.
 30. A method according to claim 29, wherein couplingof said magnetic sensor and said object and coupling of said object andsaid magnetic particles, are carried out at the same time in a reactionvessel containing said magnetic sensor.
 31. A method of assaying anobject using a biosensor according to claim 2, in which said magneticparticles can replace an object bound to said magnetic sensorreversibly, wherein said method comprises the steps of: determining anamount of said magnetic particles which have bound to said magneticsensor by replacement of said object using said biosensor; anddetermining an amount of said object based on the amount of saidmagnetic particles.